ABSTRACTBreast cancer remains a world-wide challenge, and additional anti-cancer therapies are still urgently needed. Emerging evidence has demonstrated the potent anti-tumor effect of biguanides, among which phenformin was reported to potentially be a more active anti-cancer agent than metformin. However, little attention has been given to the role of phenformin in breast cancer. In this study, we reveal the role of phenformin in cell death of the MCF7, ZR-75-1, MDA-MB-231 and SUM1315 breast cancer cell lines. The respective IC50 values of phenformin in MCF7, ZR-75-1, MDA-MB-231 and SUM1315 cells were 1.184±0.045 mM, 0.665±0.007 mM, 2.347±0.010 mM and 1.885±0.015 mM (mean± standard error). Phenformin induced cell cycle change and apoptosis in breast cancer cells via the AMPK/mTOR/p70s6k and MAPK/ERK pathways. Interestingly, phenformin induced MET (mesenchymal-epithelial transition) and decreased the migration rate in breast cancer cell lines. Furthermore, our results suggest that phenformin inhibits breast cancer cell metastasis after intracardiac injection into nude mice. Taken together, our study further confirms the potential benefit of phenformin in breast cancer treatment and provides novel mechanistic insight into its anti-cancer activity in breast cancer.

pone.0131207.g008: Phenformin inhibits the metastasis of MDA-MB-231 cells in nude mice.(A) The luciferase-tagged MDA-MB-231 cells were inoculated intracardially into female nude mice. After the injections, the mice were separated into the control and phenformin groups (10 mice per group). The control group’s drinking water was replaced with 5% sucrose. The phenformin group’s drinking water was replaced with 5% sucrose containing phenformin (300 mgkg-1). The development of metastasis was monitored using whole mouse fluorescence and bioluminescence imaging(negative control: mice did not receive an intracardiac injection of luciferase-expressing cells). (B)Four weeks after the intracardiac injections, the total flux signals in the control group were significantly higher than those in the phenformin group (p = 0.0065). The data are presented as the mean±SEM and the Wilcoxon rank sum test was used to identify significant differences in total flux between the control and phenformin- treated animals.

Mentions:
Phenformin treatment significantly increased the expression of the epithelial marker E-cadherin in MDA-MB-231 cells. Moderate changes in E-cadherin expression were observed in MCF-7, ZR-75-1 and SUM1315 cells. In contrast, the mesenchymal marker vimentin was significantly downregulated in all the cell lines after treatment with phenformin (Fig 6). Our findings indicated that phenformin might induce MET and prevent breast cancer metastasis. As the MDA-MB-231 cell line is the least sensitive of the four cell lines to phenformin-induced growth inhibition and the most aggressive among the four breast cancer cell lines, we utilized MDA-MB-231 cells to further evaluate the role of phenformin in breast cancer metastasis. Interestingly, the migration rate of phenformin-treated MDA-MB-231 cells was significantly decreased compared with control cells (Fig 7). Furthermore, four weeks after intracardiac injection of MDA-MB-231 cells, the total flux of the nude mice in the phenformin group was significantly lower than that in the control group (p = 0.0065) (Fig 8), which indicated that phenformin might inhibit breast cancer metastasis.

pone.0131207.g008: Phenformin inhibits the metastasis of MDA-MB-231 cells in nude mice.(A) The luciferase-tagged MDA-MB-231 cells were inoculated intracardially into female nude mice. After the injections, the mice were separated into the control and phenformin groups (10 mice per group). The control group’s drinking water was replaced with 5% sucrose. The phenformin group’s drinking water was replaced with 5% sucrose containing phenformin (300 mgkg-1). The development of metastasis was monitored using whole mouse fluorescence and bioluminescence imaging(negative control: mice did not receive an intracardiac injection of luciferase-expressing cells). (B)Four weeks after the intracardiac injections, the total flux signals in the control group were significantly higher than those in the phenformin group (p = 0.0065). The data are presented as the mean±SEM and the Wilcoxon rank sum test was used to identify significant differences in total flux between the control and phenformin- treated animals.

Mentions:
Phenformin treatment significantly increased the expression of the epithelial marker E-cadherin in MDA-MB-231 cells. Moderate changes in E-cadherin expression were observed in MCF-7, ZR-75-1 and SUM1315 cells. In contrast, the mesenchymal marker vimentin was significantly downregulated in all the cell lines after treatment with phenformin (Fig 6). Our findings indicated that phenformin might induce MET and prevent breast cancer metastasis. As the MDA-MB-231 cell line is the least sensitive of the four cell lines to phenformin-induced growth inhibition and the most aggressive among the four breast cancer cell lines, we utilized MDA-MB-231 cells to further evaluate the role of phenformin in breast cancer metastasis. Interestingly, the migration rate of phenformin-treated MDA-MB-231 cells was significantly decreased compared with control cells (Fig 7). Furthermore, four weeks after intracardiac injection of MDA-MB-231 cells, the total flux of the nude mice in the phenformin group was significantly lower than that in the control group (p = 0.0065) (Fig 8), which indicated that phenformin might inhibit breast cancer metastasis.

Bottom Line:
Emerging evidence has demonstrated the potent anti-tumor effect of biguanides, among which phenformin was reported to potentially be a more active anti-cancer agent than metformin.Furthermore, our results suggest that phenformin inhibits breast cancer cell metastasis after intracardiac injection into nude mice.Taken together, our study further confirms the potential benefit of phenformin in breast cancer treatment and provides novel mechanistic insight into its anti-cancer activity in breast cancer.

ABSTRACTBreast cancer remains a world-wide challenge, and additional anti-cancer therapies are still urgently needed. Emerging evidence has demonstrated the potent anti-tumor effect of biguanides, among which phenformin was reported to potentially be a more active anti-cancer agent than metformin. However, little attention has been given to the role of phenformin in breast cancer. In this study, we reveal the role of phenformin in cell death of the MCF7, ZR-75-1, MDA-MB-231 and SUM1315 breast cancer cell lines. The respective IC50 values of phenformin in MCF7, ZR-75-1, MDA-MB-231 and SUM1315 cells were 1.184±0.045 mM, 0.665±0.007 mM, 2.347±0.010 mM and 1.885±0.015 mM (mean± standard error). Phenformin induced cell cycle change and apoptosis in breast cancer cells via the AMPK/mTOR/p70s6k and MAPK/ERK pathways. Interestingly, phenformin induced MET (mesenchymal-epithelial transition) and decreased the migration rate in breast cancer cell lines. Furthermore, our results suggest that phenformin inhibits breast cancer cell metastasis after intracardiac injection into nude mice. Taken together, our study further confirms the potential benefit of phenformin in breast cancer treatment and provides novel mechanistic insight into its anti-cancer activity in breast cancer.